PONE-D-20-05825

Utilizing Methylglyoxal and D-lactate in Urine to Evaluate Saikosaponin C Treatment in Mice with Accelerated Nephrotoxic Serum Nephritis

PLOS ONE

Dear Doctor Shih-Ming Chen,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

After careful reading I think that the paper is of interest. However, some major points have been raised by the reviewers. Please answer point by point all the following issues before reconsidering your manuscript.

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Christos E. Chadjichristos

Academic Editor

PLOS ONE

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Additional Editor Comments (if provided):

After careful reading I think that the paper is of interest. However, some major points have been raised by the reviewers. Please answer point by point all the following points before reconsidering your manuscript.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Chia-Yu Lin et al are interested on Saikosaponin (SSC) a bioactive element of the medicinal plant Bupleurum falcatum L, largely used in traditional medicine in Asia for the treatment of various diseases. Here they investigate the effect of three SSC doses, on mice with accelerated nephrotoxic serum nephritis (NTS) as well as the relationship between Methylglyoxal (MGO) and D-lactate, two compounds known to be related to renal injury. Indeed, the authors demonstrate that urinary MGO and D-lactate levels correlate with renal damages.

Although the SSC treatment does not completely prevent disease development, they convincingly show the effect of an intermediate dose of SSC in delaying disease progression. However, not all the aspects of the manuscripts are sufficiently developed and some conclusions are not supported by data, or further investigated. In addition, some clarifications are needed, in particular with regards to the relationship between MGO and L-lactate, there is no evidence of any direct link between them.

Nevertheless, the study remains interesting, but the authors should improve their characterization and perform more analysis. Overall, the validation of the presented results with more data would improve the relevance of the manuscript.

Major comments

- It is somehow counterintuitive that only the intermediate, but not the high dose, of SSC is protective. This should be discussed. Potential toxic effect of the high dose should be evaluated in more detail (i.e. biochemical analysis or body weight).

- The authors state that the intermediate dose decreases the inflammatory immune infiltration and inflammation. However, these data are not shown and/or quantified.

- Mechanistic insights

Figure2: Are urine collected at the end-time point? What about proteinuria at day 15 (end of experiment)?

Figure 3-Figure 4

The authors describe NTS induced-damage with and histological score (defined as cell proliferation, thrombus, and crescent formation) and area of fibrosis. It would be interesting to have more information about the histological differences in mice. Are any of the analysed parameters selectively improved by the treatment with SSC? What about tubular or glomerular damage? As I have already pointed out, the authors mention differences in the inflammatory process but this is not support by any data/quantification. Staining for major immune populations should be done.

Figure5-6: Same as in figure 2. Did the authors measure these urinary parameters at end-point (day +15)?

"The appropriate dosage of saikosaponin C treatment may improve the immune-reaction and down-regulate methylglyoxal and D-lactate production"

In the discussion the authors explain the anti-inflammatory effect of SSC may be related to glucocorticoid receptor (GR) activation and inhibition of glycolysis. This is a hasty conclusion that can be true but this is not supported by any data/quantification for anti-inflammatory effect or any link with GR.

Figure7:

The WB blot shown for glyoxalase 1(GLOT1) is not representative of the quantification provided. Glot1 seems more expressed in SC group compared to the N group. The GLOT1 expression looks similar in the M and H grouped. Was GLOT1 expression evaluated in all the treated mice? Additional WB image should be provided.

Minor comment

I would suggest to display individual data points in graphs (dot plot).

Reviewer #2: The paper presented by Lin et al aims to evaluate the effect of different dosages of SSC on mice with accelerated NTS nephritis, and the relationship between urinary MGO and D-lactate during SSC treatment of NTS nephritis.

The paper is simple to read, written almost good English. Data are generally presented clearly, and the analyses well described and reported in the text. I propose some major revisions to make the paper suitable for publication in Plos One.

- The images must absolutely re edited clearly. The colors are too strong and the contrast difficult to understand (masson staining has the same colors than H & E!).

- I suggest the evaluation of fibrosis using Sirius red staining.

- The clear explanation of the histological score is mandatory!

- As correctly reported by the authors, probably the degree of proteinuria reflects a podocyte damage. In order to evaluate it and increase the power of the result presented I suggest the evaluation of podocyte marker (f.i. nephrin, synaptopodin) in immunofluorescence of (better) in PCR.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-20-05825R1

Utilizing Methylglyoxal and D-lactate in Urine to Evaluate Saikosaponin C Treatment in Mice with Accelerated Nephrotoxic Serum Nephritis

PLOS ONE

Dear Dr. Shih-Ming Chen,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Indeed, after careful reading and based also to the reviewer's suggestions, I think that before publication two issues have to be addressed. Please provide:

1/  immunostainings for inflammatory cells, and

2/  scale bars for all pictures

Please submit your revised manuscript by October 9th 2020. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Christos E. Chadjichristos

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Overall, the authors have addressed the points raised by the reviewers and I appreciate their effort. However, few responses are not entirely convincing/comprehensive.

The potential toxicity of the high dose is discussed, but no data shown to support their statement. An “immune-related influence” is suggested but, again, no supporting data.

The inflammatory infiltrate quantification is now provided based on a histological score. Specific immunostaining of inflammatory cells would have been preferable and more representative.

The authors are also showing a reduction in inflammation by measuring TNF. Why did they only measure TNF?

Figure 3-4 and 5 please ensure that the magnification is the same for all panels. Scale barre are not provided for figure.

WB in Figure 7. I would have preferred to see the individual mice, not to pool them.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Utilizing Methylglyoxal and D-lactate in Urine to Evaluate Saikosaponin C Treatment in Mice with Accelerated Nephrotoxic Serum Nephritis

PONE-D-20-05825R2

Dear Dr. Shih-Ming Chen,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Christos E. Chadjichristos

Academic Editor

PLOS ONE